H. pylori is a gram-negative and microaerophilic bacterium, which causes chronic gastritis. It is estimated that about half of the world's population are infected with this bacterium. Most of the H. pylori infected individuals are asymptomatic despite gastritis but a minority will develop severe sequelae. H. pylori is the key factor in the development of duodenal or gastric ulcers, and the most common single risk factor for non-cardia gastric cancer. The successful eradication of H. pylori infection leads to healing of peptic ulcer disease (Ford et al, 2003) and in long-term relief of dyspeptic symptoms even in some patients without ulcers (Ford et al., 2004). Eradication therapy of H. pylori infection is also recommended as the first-line treatment for low-grade mucosa-associated lymphoid tissue (MALT) lymphoma and on the basis of epidemiological studies successful H. pylori eradication therapy may lead to a decreased number of gastric cancer cases (Kosunen et al, 2011; Chey and Wong, 2007).
Diagnostic methods for the detection of H. pylori infection can be divided into invasive (gastroscopy is required) and non-invasive methods. Although several methods give highly accurate results, there is not a single gold standard for the diagnosis of H. pylori infection but the selection of the methods depends on the clinical situation and if there is otherwise a need for gastroscopy. According to the test-and treat strategy, patients with a low risk for gastric cancer can be tested for H. pylori with non-invasive methods and treated if H. pylori is detected.
Although non-invasive diagnostic methods, such as urea breath tests and stool antigen tests, in general show highly accurate detection rates, these methods do not give any information on the antimicrobial susceptibility of the infecting isolate of H. pylori. Furthermore, even if gastric biopsies are taken in gastroscopy and sent for culture, antimicrobial susceptibility testing results are usually not available in all positive cases due to the low sensitivity of culture. However, the need for antimicrobial susceptibility testing of H. pylori is increasing. Eradication therapy of H. pylori usually consists of two different antimicrobial agents and a proton pump inhibitor. Clarithromycin is an important component of the classical triple therapy and in the case of clarithromycin resistant H. pylori, the clarithromycin-based regimen results in eradication failure in the vast majority of the cases (Fischbach and Evans, 2007).
Due to the increasing clarithromycin resistance rates (Malfertheiner et al., 2012) the latest European guidelines recommend the use of molecular tests in the detection of H. pylori in gastric biopsies and antimicrobial susceptibility testing if culture is not available. Molecular methods have been developed for the detection of H. pylori and the simultaneous testing for clarithromycin susceptibility of the isolates. Clarithromycin resistance of H. pylori is well known and due to point mutations within the peptidyltransferase region of the 23S rRNA gene.
The high clinical relevance of H. pylori infection in gastric mucosa has stimulated the development of several PCR based diagnostic methods detecting H. pylori DNA in stool samples. However, the problem of low sensitivity has frequently arisen. These results may have been due to a lack of intact H. pylori DNA in stools. In contrast to intestinal bacterial pathogens, which are found in viable form at high concentrations in stools, living H. pylori is most likely not present at all and, consequently, its DNA may be present only in a degraded form rendering the detection more challenging. Although some PCR-based methods have shown accurate results in the detection of H. pylori and testing of clarithromycin susceptibility in gastric biopsies, problems have arisen when the methods have been used in stool samples. Due to some major limitations, such as PCR inhibitors (Monteneiro, 1997) and low concentrations of mostly fragmented H. pylori DNA in fecal samples, it has been difficult to develop methods sensitive enough. The PCR-based methods have only shown sensitivities about 60% when applied for stool samples (Lottspeich, 2007 etc). The purpose of the present study was to develop a highly accurate non-invasive method for detection of H. pylori and concomitant clarithromycin susceptibility of the isolate in fecal samples.
Schabereiter-Gurtner et al. (2004) disclosed a real-time PCR assay for detection of H. pylori infection and simultaneous clarithromycin susceptibility testing in stool samples. In practice, the authors detected point mutations in the 23S rRNA gene of H. pylori associated with clarithromycin resistance. However, Lottspeich et al. (2007) evaluated the method and concluded that detection of H. pylori DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA for the accurate diagnosis of H. pylori infection. Later, Scaletsky et al. (2011) found that the method proved to be appropriate for H. pylori clarithromycin susceptibility testing, although the possibility of missing some positive results should be taken into account. Other publications disclosing primers and/or probes specific to H. pylori 23S rRNA gene are: Fontana et al., 2003; Noguchi et al., 2007; Dewhirst et al., 2005; Maeda et al., 1998; Khan et al., 2004; and Rimbara et al., 2005.
PCR assays for detection of H. pylori directed to other target genes than the 23S rRNA gene are disclosed in the following publications: Falsafi et al., 2009; Singh et al., 2008; Monteiro et al., 2001; Makristathis et al., 1998; Mishra et al, 2008; and Burucoa et al., 1999.
In the development of PCR assays, one of the most important factors is to locate oligonucleotide sequences that enable reliable species-specific amplification, detection and quantification. It is of utmost importance that a given set of oligonucleotides, designed to amplify H. pylori, does not cross-react with DNA originating from any other species possibly present in a sample. Finding such sequences can be far from trivial, at least for the following reasons: 1) Many of the species are relatively closely related, making it challenging to locate sequences that are unique for each species; 2) Pathogen strains originating from a single species can be genetically diverged, making it difficult to locate sequences that would enable equally efficient amplification of all strains within a species; 3) The sample may contain PCR inhibitors or as in this case the sample contains mainly fragmented target DNA, since H. pylori typically thrives in gastric mucosa and will very likely die and deteriorate in large intestine. Hence, effective amplification of pathogen DNA from a stool sample requires oligonucleotide design enabling high PCR efficiency (optimally as close to 100% as possible).
Compared to the prior art, the present invention provides at least the following major advantages: the difference of Tm of outer and inner primers is optimized to achieve simpler reaction routines, e.g. the nested PCR reaction of the present invention can be performed in a single vessel. Rimbara et al, 2005, and Noguchi et al., 2007, disclose methods where nested PCR is performed sequentially in two separate reactions. The robustness of the PCR reaction of the present invention is also on the level that there is no need to isolate and purify the DNA from a stool sample by phenol extraction or sample homogenization as is done in Rimbara et al, 2005, and Noguchi et al., 2007, respectively. Furthermore, the length of the amplicon which is amplified in the second reaction of the nested PCR in Rimbara et al, 2005, and Noguchi et al., 2007, is too long to be sensitively detected in real-time PCR. The length of the amplicon amplified by the inner primers of the present invention is 143 bp while in Rimbara it is 463 bp and in Noguchi it is 367 bp.
The disclosures by Fontana et al., 2003, JP 2005168474, and Booka et al., 2005 are not directed to a nested PCR reaction and consequently the primers disclosed are not compatible with nested reactions without modification. Further, the length of the amplicon amplified in Fontana et al., 2003, is 991 bp and is thus too long to be detected in real time PCR. It is also noteworthy that Fontana et al. are amplifying a different region of 23S rRNA gene than Noguchi et al. Therefore, it is clear that the prior art is teaching that there are alternative regions in 23S rRNA which are suitable as target regions for a PCR based detection of the presence of Helicobacter strains. In JP 2005168474, the primers seem not to be specific to Helicobacter but may also cross-react with Campylobacter strains.
Although numerous PCR based assays for detecting H. pylori are already disclosed, there is still a need in the field for a PCR assay which is able to provide high specificity and reliability for the detection. The present inventors have now located DNA sequence regions in H. pylori 23S rRNA gene that are surprisingly well-suited for specific and sensitive amplification of H. pylori DNA from stool. Optimal primers and quantitative PCR probes have been designed and validated for identification of the presence of H. pylori in patients.